Molecular characterization of Listeria spp. isolated from cattle farms, abattoirs, and beef products in Mpumalanga and North-West Provinces, South Africa

Abstract

Listeria monocytogenes is a foodborne pathogen that has serious public health implications. Since the advent of the largest outbreak of listeriosis in South Africa, it has become important to understand the genomic characteristics of L. monocytogenes from food products. This study aimed to use molecular techniques to characterize Listeria isolates (n=214) recovered from cattle farms, beef and beef-based products from retails. PCR was used to classify the isolates into Listeria species. The identified L. monocytogenes were further classified into serogroups and Multilocus Variable-Number Tandem Repeat Analysis (MLVA) types using conventional PCR protocols. Likewise, L. innocua (165) isolates were also typed using MLVA. As the only pathogenic species identified in this study, L. monocytogenes was further characterized using whole genome sequencing (WGS) and bioinformatics tools to determine the population structure, antimicrobial resistance genes, virulence profile, mobile genetic elements (plasmids, prophages), genomic islands, insertion sequences, the type VII secretion system, and sortases. Listeria isolates were classified into L. innocua (77.10%, n=165), L. monocytogenes (11.21%, n=24), L. welshimeri (5.61%, n=12), L. grayi (1.40%, n=3), L. seeligeri (0.93%, n=2), and L. species (3.73%, n=8). L. monocytogenes serogroups determined by PCR were: IVb (4b-4d-4e) (37.50%), IIa (1/2a-3a) (29.16%), IIb (1/2b-3b) (12.50%), IIc (1/2c-3c) (8.33%), and IVb-1 (4.16%). MLVA was able to cluster L. monocytogenes isolates into 10 MLVA types and L. innocua into 34 MLVA types based on their relatedness. The isolates clustered irrespective of sample category, geographical origin and serogroup for L. monocytogenes. Multilocus Sequence Type (MLST) analysis revealed that ST204 of CC204 (Lineage II, serogroup IIa (1/2a, 3a) was the most common sequence type (ST). Other sequence types detected included ST1 of CC1 (Lineage I, serogroups IVb (4b,4d,4e), ST5 of CC5 (Lineage I, serogroup IIb (1/2b,3b,7), ST9 of CC9 (Lineage II, serogroup IIc (2c, 3c), ST88 of CC88 (Lineage I, serogroup IIb (1/2b,3b,7), ST876 of CC1, (Lineage II, serogroup IVb (4b,4d,4e), ST2 of CC2 (Lineage I, serogroup IVb (4b, 4d, 4e), ST321 of CC321 (Lineage II, serogroup IIa (1/2a, 3a) and ST1430 of CC2 (Lineage I, serogroup IVb (4b,4d,4e). All the L. monocytogenes STs carried four intrinsic resistance genes, fosX, lin, norB, and mprF, conferring resistance to antimicrobials, fosfomycin, lincosamide, quinolones, and cationic peptides, respectively. Genes encoding for virulence factors LIPI-1 (pfrA-hly-plcA-plcB-mpl-ActA) and internalin genes inlABCJKF, were present in most STs. Prophages profile, vB_LmoS_188, was overrepresented amongst the isolates, followed by LP_101, LmoS_293_028989, LP_030_2_021539, A006 and LP_HM00113468. Plasmid pLGUG1 (40%) was the most represented and only found in ST204 types. Similarly, plasmid J1776 (40%) was also significantly represented amongst the STs, followed by pLI100 (13%), and pLM5578 (7%). Mobile genetic elements did not harbour any virulence or resistance genes. Listeria genomic island 2 (LGI-2) was found present in all the isolates, whilst Listeria genomic island 3 (LGI-3) was present in a subset of isolates (25%). The type VII secretion system was found in 42% of the isolates, and sortase A was found in all L. monocytogenes genomes. This study revealed that non-pathogenic and pathogenic Listeria spp. could be contaminants of meat products and the farm environment. The strains of L. innocua and L. monocytogenes displayed diversity even though they all originated from bovine samples. MLVA proved to be an affordable, simple, and discriminatory method that can be used routinely to type L. monocytogenes and L. innocua isolates. The isolates did not carry genes conferring resistance to first-line drugs used against listeriosis and, therefore, did not pose a threat to antimicrobial therapy. Characterization of L. monocytogenes in the current study highlighted the virulence capability of L. monocytogenes and the risk posed to the public by this pathogen, as it is often found in food and food processing environments.